The present invention relates to antioxidant peptides and methods for isolating antioxidative peptides. More specifically, the present invention relates to antioxidant peptides derived from casein. These antioxidant peptides may be used as food supplements or food additives.
Oxidation of oils and fats in foods causes deterioration of flavor and degradation of oil/fat quality. Furthermore, the intake of peroxides produced form lipid oxidation exerts detrimental effects in vivo. Free radicals and active oxygen species generated in the course of oxidation reactions denature proteins in vivo, inactivate enzymes (Szweda et al., xe2x80x9cInactivation of glucose-6-phosphate dehydrogenase, by 4-hydroxy-2-nonene modification of an active-site lysine,xe2x80x9d J. Biol. Chem. 268:3342 (1993)), bring about mutations in DNA (Reiss et al., xe2x80x9cDNA-malonaldehyde reaction: Formation of fluorescent products,xe2x80x9d Biochem. Biophys. Res. Commun. 48:921 (1972)), modify low density lipoproteins (Alaiz et al., xe2x80x9cModification of delipidated apoprotein B of low density lipoprotein by lipid oxidation products in relation to macrophage scavenger receptor binding,xe2x80x9d Biol. Phar. Bull. 17:51 (1994)), and contribute to aging and various diseases such as cancer. Dietary antioxidants may help prevent cardiovascular diseases (Krinksy, N. I., xe2x80x9cAction of carotenoids in biological systems,xe2x80x9d Annu. Rev. Nutri. 13:561 (1993); Parthasarathy, S., xe2x80x9cMechanisms by which dietary antioxidants may prevent cardioscular diseases,xe2x80x9d J. Med. Food 1:45 (1998)).
Many substances have been identified which have antioxidative activity. These include glutathione, carnosine (Zhou et al., xe2x80x9cAbility of carnosine and other skeletal muscle components to quench unsaturated aldehydic lipid oxidation products,xe2x80x9d J. Agric. Food Chem. 47:51(1999)), certain amino acids (Marcuse et al., Nature, (1960)), certain proteins, including lactoferrin (Gutteridge et al., Bioch. J. 199:259 (1981)), casein (Laakso, xe2x80x9cInhibition of lipid peroxidation by casein. Evidence of molecular encapsulation of 1,4-pentadiene fatty acids,xe2x80x9d Biochim. Biophys. Acta 792:11 (1984)), and certain peptides (Tomita et al., xe2x80x9cAntioxidant,xe2x80x9d U.S. Pat. No. 5,804,555 (1998), Suetsuna et al., xe2x80x9cIsolation and characterization of free radical scavenging activities peptides derived from casein,xe2x80x9d J. Nurt. Biochem. 11:128 (2000)). However, because of the importance in preventing oxidation to biological processes and to improved stability of products subject to oxidation, there remains a need to identify new antioxidative compounds, such as antioxidant peptides. Furthermore, there remains a need for a general method that can be used to isolate antioxidative peptides from virtually any protein source.
Miclo et al, U.S. Pat. No. 5,846,939, described a decapeptide from alpha s1 casein with benzodiazepine-type activity which is useful for the treatment of convulsions and anxiety. However, Miclo et al. did not analyze the decapeptide, or fragments thereof, for antioxidative activity.
Shimamura et al., U.S. Pat. No. 5,952,193, described a method for producing a peptide mixture from whey protein utilizing hydrolysis carried out by a protease. In some embodiments, the peptides generated by hydrolysis are further purified. However, Shimamura et al., did not disclose phase separation of the peptides nor determine antioxidative activities of the peptide mixture.
Tomita et al., U.S. Pat. No. 5,804,555, described an antioxidant hydrolysate of lactoferrin. After cleaving lactoferrin with protease, the resulting peptides were purified by reverse phase HPLC. Tomita et al. did not disclose a method for isolating antioxidative peptide fractions that utilizes a separation step that does not result in purified peptide fractions, or that is used in combination with, or in place of, reverse phase HPLC or size-based separation. Additionally, Tomita et al. did not disclose antioxidative peptides of casein, whey, or soy protein. Finally, Tomita et al. did not disclose a phase separation step in isolating antioxidative peptide fragments.
Suestsuna et al. (xe2x80x9cIsolation and characterization of free radical scavenging activities peptides [sic] derived from casein,xe2x80x9d J. Nutr. Biochem., 11:128 (2000)) described peptides with antioxidative activity generated from proteolytic cleavage of casein. Peptides with sequence EL, YFYPEL, FYPEL, YPEL, and PEL were provided. The peptides were purified using a method including several column chromatography-steps.
Thus, there remains a need for additional anti-oxidative peptides and for easier to use, and more cost effective, methods for isolating anti-oxidative peptides. Additionally, there remains a need for a general method of anti-oxidative peptide isolation that can be utilized to isolate peptides from many protein sources.
The current invention provides antioxidative peptides from casein. Furthermore, the current invention includes methods that can be used to isolate antioxidative peptide fractions or antioxidative peptides from any protein source.
The current invention provides antioxidative peptide fractions. The invention includes antioxidative peptides comprising sequences SEQ ID NOS:1-5. The invention includes methods for making the antioxidative peptides, isolated nucleic acids encoding the antioxidative peptides, and expression vectors comprising these nucleic acids. The invention includes food additives for preventing oxidation in vivo. The invention includes stabilized products with improved storage characteristics which are oxidation resistant. Finally, the invention includes methods for isolating peptide fractions having antioxidative activity from protein samples.
In one aspect, the present invention is an isolated peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein the peptide has antioxidative activity. In one embodiment, the amino acid sequence is SEQ ID NO:1. In another embodiment, the amino acid sequence is SEQ ID NO:3. In another embodiment, the amino acid sequence is SEQ ID NO:4. In another embodiment, the amino acid sequence is SEQ ID NO:5.
In one aspect, the present invention is an isolated antioxidative casein peptide consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In one embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:1. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:2. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:3. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:4. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:5.
In another embodiment, the isolated antioxidative casein peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In one embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:1. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:2. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:3. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:4. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:5.
In another aspect, the current invention is a food supplement comprising: an antioxidative peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NQ.1, SEQ IN DO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, said antioxidative peptide being present in an amount effective for preventing in vivo oxidation; and an orally-ingestible diluent or carrier. In one embodiment of this aspect of the invention, the amino acid sequence is SEQ ID NO:1. In another embodiment, the amino acid sequence is SEQ ID NO.3. In another embodiment, the amino acid sequence is SEQ ID NO:4. In another embodiment, the amino acid sequence is SEQ ID NO:5.
In another embodiment of this aspect of the invention directed to a food supplement, the antioxidative peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In one embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:1. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:2. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:3. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:4. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:5.
In another aspect, the current invention is a stabilized product comprising: a product ingredient subject to oxidation; and an antioxidative peptide selected from the group consisting essentially of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein the antioxidative peptide is present in an amount effective for preventing oxidation of the product ingredient. In one embodiment of this aspect of the invention directed to a stabilized product, the antioxidative polypeptide consists essentially of the amino acid sequence of SEQ ID NO:1. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:2. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:3. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:4. In another aspect, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:5.
In another embodiment of this aspect of the invention directed to a stabilized product, the antioxidative peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In one embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:1. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:2. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:3. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:4. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:5.
In one embodiment of this aspect of the invention, the product ingredient subject to oxidation is a food ingredient and the product is a food. In another embodiment, the product ingredient subject to oxidation is a medical diagnostic reagent component and the stabilized product is a medical diagnostic reagent. In another embodiment, the product ingredient subject to oxidation is a pharmaceutical and the stabilized product is a pharmaceutical product.
In another aspect, the current invention is an antioxidative therapeutic compound for treating a disease involving oxidation, wherein the therapeutic compound comprises: an antioxidative peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ IN DO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, wherein the antioxidative peptide has antioxidative activity, and wherein the antioxidative peptide is present in an amount effective for preventing in vivo oxidation; and a pharmaceutically acceptable carrier. In one embodiment of this aspect of the invention directed to a compound for treating a disease involving oxidation, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:1. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:2. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:3. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:4. In another embodiment, the antioxidative peptide consists essentially of the amino acid sequence of SEQ ID NO:5.
In another embodiment of this aspect of the invention directed to a therapeutic compound for treating a disease involving oxidation, the antioxidative peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In one embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:1. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:2. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:3. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:4. In another embodiment, the antioxidative peptide consists of the amino acid sequence of SEQ ID NO:5.
In another aspect, the current invention provides a method for treating a condition associated with oxidation in a subject, said method comprises administering to the subject an amount of an antioxidative therapeutic compound as described herein. In one embodiment of this aspect of the invention directed to a method for treating a condition associated with oxidation, the subject is a mammal, including a human. In another embodiment of this aspect of the invention directed to a method for treating a condition associated with oxidation, the condition is selected from an inflammatory condition, an allergic condition, and an auto-immune condition. In another embodiment of this aspect of the current invention, the condition associated with oxidation is selected from osteoarthritis, rheumatoid arthritis, ischemia, cataract, corneal pathology, glaucoma, retinal degeneration, vitreal degeneration, cancer, immune deficiency, hyperimmunity, autoimmunity, neurodegeneration, aging, Alzheimer""s disease, Huntington""s disease, Machoado-Joseph disease, multiple sclerosis, muscular dystrophy, Parkinson""s disease, senility, muscular atrophy, stroke, hepatopathies, systemic lupus erythematosus, mixed connective tissue disease, multiple sclerosis, and diabetes.
In another embodiment, the current invention is directed to an isolated nucleic acid encoding an antioxidative peptide, wherein the nucleic acid consists essentially of a nucleotide sequence that encodes the antioxidative peptide having a sequence of the peptide sequences listed as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
In one embodiment of this aspect of the invention directed to a nucleic acid encoding an antioxidative peptide, the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:1. In another embodiment, the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:2. In another embodiment, the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:3. In another embodiment, the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:4. In another embodiment, the isolated nucleic acid encodes the amino acid sequence of SEQ ID NO:5.
In one embodiment of this aspect of the invention directed to a nucleic acid encoding an antioxidative peptide, the isolated nucleic acid consists essentially of the nucleotide sequence of SEQ ID NO:6. In another embodiment, the isolated nucleic acid consists essentially of the nucleotide sequence of SEQ ID NO:7. In another embodiment, the isolated nucleic acid consists essentially of the nucleotide sequence of SEQ ID NO:8. In another embodiment, the isolated nucleic acid consists essentially of the nucleotide sequence of SEQ ID NO:9. In another embodiment, the isolated nucleic acid consists essentially of the nucleotide sequence of SEQ ID NO:10.
In one embodiment of this aspect of the invention directed to a nucleic acid encoding an antioxidative peptide, the isolated nucleic acid consists of the nucleotide sequence of SEQ ID NO:6. In another embodiment, the isolated nucleic acid consists of the nucleotide sequence of SEQ ID NO:7. In another embodiment, the isolated nucleic acid consists of the nucleotide sequence of SEQ ID NO:8. In another embodiment, the isolated nucleic acid consists of the nucleotide sequence of SEQ ID NO:9. In another embodiment, the isolated nucleic acid consists essentially of the nucleotide sequence of SEQ ID NO:10.
In another aspect, the current invention provides a recombinant expression vector that expresses an antioxidative peptide, said vector comprising a promoter linked to an antioxidative peptide encoding nucleic acid. The antioxidative peptide encoding nucleic acids useful for this aspect of the invention are those described above.
In another embodiment, the current invention provides a host cell transformed with an antioxidative peptide encoding nucleic acid, as described above. In preferred embodiments, the host cell is transformed with a recombinant expression vector that expresses an antioxidative peptide, as described above.
In another aspect, the current invention provides a method for producing an antioxidative casein peptide in a host cell, said method comprising the steps of:
(a) introducing a nucleic acid encoding an antioxidative peptide having an amino acid sequence selected from SEQ ID NOS:1-5 into a vector, thereby producing an antioxidative casein peptide expression vector;
(b) introducing the antioxidative casein peptide expression vector into the host cell to produce an engineered host cell;
(c) maintaining the engineering host cell under conditions suitable for the expression of an antioxidative casein peptide by the host cell; and
(d) collecting the antioxidative casein peptide produced by the host cell.
In preferred embodiments, the host cell is transformed with a recombinant expression vector that expresses an antioxidative peptide, as described above.
In another aspect, the current invention includes methods for identifying antioxidative peptides and fractions containing these peptides. This aspect of the invention includes peptides whose sequence is determined by the methods for identifying antioxidative peptides. The methods of this aspect of the invention utilize a separation step that does not involve column chromatography, to form at least two fractions. In certain embodiments, the separating does not involve substantially purifying any of the peptides.
One embodiment of this aspect of the invention provides a method for identifying an antioxidative peptide from a mixture of peptides, said method comprising:
(a) generating a mixture of peptides suspected of containing an antioxidative peptide or peptides;
(b) separating the peptides in the mixture based on charge or hydrophobicity, using a technique other than column chromatography, to form at least two fractions;
(c) assessing the fractions for antioxidative activity; and
(d) treating fractions having antioxidative activity in order to substantially purify the antioxidative peptide or peptides having antioxidative activity.
Another embodiment of this aspect of the invention comprises a method for identifying digested protein fractions containing antioxidative peptides, said method comprising:
(a) digesting a protein with a protease to produce a mixture of peptides;
(b) separating the mixture of peptides by phase separation to produce at least a first phase and a second phase;
(c) assaying the first phase and the second phase for antioxidative activity to determine whether the first phase or the second phase contain antioxidative peptides.
In one preferred embodiment, the method further comprises:
(d) isolating the phases with antioxidative activity; and
(e) substantially purifying the antioxidative peptide or peptides in the phases having antioxidative activity.
In another embodiment of this aspect of the invention, the current invention is an antioxidative peptide obtained by the following process:
(a) providing a mixture of peptides suspected of containing an antioxidative peptide or peptides and other macromolecules;
(b) separating the peptides in the mixture based on charge or hydrophobicity, using a technique other than column chromatography, to form at least a first fraction and a second fraction;
(c) assessing the first fraction and the second fraction for antioxidative activity;
(d) isolating the fractions having antioxidative activity; and
(e) substantially purifying the antioxidative peptide or peptides in the fractions having antioxidative activity, thereby isolating the antioxidative peptide or peptides.
In certain embodiments, the method further comprises:
(f sequencing the antioxidative peptide or peptides.
In certain embodiments of this aspect of the invention comprising a method involving a separation step, as defined above, or a peptide identified by such a method, the step of separating comprises a step wherein peptides are not substantially purified. In a preferred embodiment, the step of separating comprises a phase separation step.
In certain embodiments of this aspect of the invention, the mixture of peptides are generated by proteolytic cleavage of milk proteins. For certain embodiments of this aspect of the invention, peptide mixtures are generated by proteolytic cleavage of proteins carried out with a protease. In certain embodiments, the protease is selected from the group consisting of glutamyl endopeptidase, corolase PN-L, papain, promod 24 L, validase actinidin, alkaline protease, flavozyme, neutral bacterial proesase Enzyco protease S-11562, Enzyco protease S-11650, Alcalase, and Neutrase. In certain embodiments, the mixture of peptides are generated by proteolytic cleavage of a mixture of proteins such as milk protein, soy protein, and whey protein. In other embodiments, the mixture of peptides are generated by proteolytic cleavage of casein.
In certain preferred embodiments, the phase separation step is carried out by adding acetone to a solution containing the mixture of peptides to form a supernatant and a precipitate. In some embodiments of this aspect of the invention, the step of separating further comprises adding ammonium sulfate to a solution containing the mixture of peptides after the phase separation step, preferably the supernatant, to form an upper layer and a lower layer, wherein the upper layer is the first fraction and the lower layer is the second fraction. In one embodiment, the fraction with antioxidative activity is the first fraction.